NLRP1 activation in human keratinocytes cultivated in organotypic skin cultures induces a psoriasis-like phenotype
EMBARGOED UNTIL 2026-03-07
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2023
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Doctoral Thesis
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EMBARGOED UNTIL 2026-03-07
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Abstract
Inflammasomes are multi-protein complexes that induce inflammation upon sensing of stress factors. They are composed of a sensor protein with different structures, such as NLRP1 (NLR (NOD-like receptor) family pyrin domain containing 1), the adaptor protein ASC and the effector protease caspase-1. When activated, the sensor induces oligomerization of ASC monomers, termed ASC specks, which activate caspase-1. Caspase-1 in turn cleaves the pro-inflammatory cytokines interleukin (IL)-1β, IL-18 and gasdermin D (GSDMD) into their mature and active forms. N-terminal GSDMD forms pores in the plasma membrane, which allow the release of IL-1β, IL-1α and IL-18, inducing inflammation, and trigger pyroptosis, a lytic form of cell death which supports inflammation.
Inflammasome activation helps to restore homeostasis when disturbed by a stressor. However, chronic inflammasome activation underlies the pathology of common inflammatory diseases. NLRP1 is the principle inflammasome sensor in human keratinocytes, but is also expressed by monocytes and neutrophils. Up to now, only genetic hints suggest a role of NLRP1 in human keratinocytes and human skin. SNPs in NLRP1 are associated with different (auto)inflammatory diseases which mainly affect the skin, such as vitiligo, atopic dermatitis, and psoriasis, and germline gain-of-function mutations in NLRP1 cause skin inflammation and predispose to the development of squamous cell carcinoma. Nevertheless, a formal proof that point to a role of NLRP1 expressed by keratinocytes in skin inflammation is missing.
NLRP1 is activated by UVB radiation, the main inducer of sunburn. As keratinocytes absorb UVB, it is believed that NLRP1 activation represents the molecular switch underlying skin inflammation in sunburn, with IL-1β being the main player. Moreover, NLRP1 is activated by dsRNA upon viral infection, the anticancer drug talabostat and 3C proteases from viruses. UVB- and dsRNA-dependent NLRP1 activation requires phosphorylation by p38 kinase.
In this thesis, we tested if activation of NLRP1 in human primary keratinocytes (HPKs) induces skin inflammation and could have a role in sunburn. As the NLRP1 pathway is not conserved in murine keratinocytes, we addressed this question by using a physiologically relevant three-dimensional (3D) fibroblast-derived matrix skin equivalent (SE) in which wild-type or HPKs lacking the expression of inflammasome components were cultivated on top of the fibroblast layer.
We detected ASC specks, which reflect inflammasome activation, exclusively in differentiated HPKs of SEs and human skin, while in 2D culture this occurs in proliferating keratinocytes. Importantly, as HPKs in monolayer highly express pro-IL-1β and secrete high amount of the active cytokine upon NLRP1 activation, NLRP1 and inflammasome activation in general has always been equated to generation of IL-1β. However, keratinocytes in human skin and SEs express very low levels of pro-IL-1β, but higher levels of pro-IL-36γ, a cytokine of the IL-1 family that induces a pro-inflammatory signature overlapping with the one induced by IL-1. This suggests that the role of keratinocyte derived IL-1β in human skin in inducing inflammation, at least at the beginning of inflammasome activation, might have been overestimated.
NLRP1 activation in keratinocytes of SEs resulted in a psoriatic phenotype, reflected by characteristic histopathological features and induction of expression of pro-inflammatory genes, including the gene encoding IL-36γ, a cytokine with a key role in psoriasis. While psoriasis has always be considered an immune-mediated inflammatory skin disease, in our model the psoriatic phenotype was induced in the absence of immune cells, pointing to an important role of keratinocytes and NLRP1 in the initial phases of psoriasis. Most importantly, ASC specks and therefore active inflammasomes were detected in suprabasal keratinocytes of psoriatic lesion. These results strongly suggest activation of NLRP1 in keratinocytes in psoriasis, most likely induced by dsRNA, which accumulates in psoriatic keratinocytes due to decreased A-to-I editing and is released upon necrosis. dsRNA-induced inflammasome activation is supported by LL-37, a cathelicidin highly expressed in psoriatic epidermis, and required phosphorylation by p38, a stress-activated kinase highly active in psoriatic epidermis.
Whereas critical roles of IL-1 and IL-36γ in psoriasis have been previously established, our results support the concept that keratinocytes are the driver and, consequently, an accessible therapeutic target for topical treatment in psoriasis. Moreover, as NLRP1 is the upstream regulator of pro-IL-1β and pro-IL-36γ, its pharmacological inhibition could block both pathways at the same time, having therefore a double function.
Interestingly, we found that prolonged NLRP1 activation or treatment with high concentrations of IL-
1 caused massive necrosis of the epidermis in SEs, most likely induced by IL-1 receptor type I (IL-1RI) activation in fibroblasts. As a similar extent of necrosis occurs in patients suffering from toxic epidermal necrolysis, a very rare and life threatening disease with incompletely understood pathophysiology, the human recombinant IL-1R antagonist Anakinra could represent a novel therapeutic option for these patients.
Surprisingly, our experiments revealed that UVB-dependent NLRP1 activation in keratinocytes of SEs is not essential for the induction of an inflammatory signature resembling sunburn. Rather, IL-1α that is released upon UVB-induced, but inflammasome-independent necrosis in SEs, induces expression of sunburn-associated cytokines and might drive sunburn in human skin as well.
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Examiner : Werner, Sabine
Examiner : Gehart, Helmuth
Examiner : Beer, Hans-Dietmar
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ETH Zurich
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Subject
Inflammasomes; inflammation; psoriasis; Keratinocytes; 3D cell culture; 3D skin model; skin equivalent; human skin
Organisational unit
02030 - Dep. Biologie / Dep. of Biology